The present invention relates to chimeric proteins with cell-targeting specificity and apoptosis-inducing activities. In particular, the invention is illustrated by a recombinant chimeric protein between human interleukin-2 (IL2) and Bax. The chimeric protein specifically targets IL2 receptor (IL2R)-expressing cells and induces cell-specific apoptosis. In accordance with the invention, chimeric proteins may be generated between any molecule that binds a specific cell type and an apoptosis-inducing protein. Such chimeric proteins are useful for selectively eliminating specific cell types in vitro and in vivo, and may be used in the treatment of autoimmunity, cancer and infectious diseases such as viral infections.
2.1. IMMUNOTOXINS
The advent of the monoclonal antibody technology and recombinant DNA technology have led to the discovery of numerous cell surface molecules associated with specific cell populations. Based on the expression pattern of these molecules, recombinant immunotoxins have been constructed to specifically target and destroy the cells that express such molecules. Recombinant immunotoxins are a class of targeted molecules designed to recognize and specifically destroy cells expressing specific receptors, such as cancer cells and cells involved in many disorders of the immune system. Generally, immunotoxins utilize a bacterial or plant toxin to destroy the unwanted cells. These molecules are designed and constructed by gene fusion techniques and are composed of both the cell targeting and cell killing moieties, a combination that makes these agents potent molecules for treatment. Examples of immunotoxins are growth factors or antigen-binding domains of antibody, including the Fv portion of an antibody (single-chain immunotoxins) fused to various mutant forms of toxin molecules. However, over the years it has become clear that treatment with such xe2x80x9cmagic bulletsxe2x80x9d for targeted immunotherapy possesses still many problems and new approaches are needed to produce improved recombinant immunotoxins.
Each recombinant immunotoxin displays some nonspecific toxicity and at sufficiently high concentrations damages normal cells that do not express the specific target antigen. This non-specific toxicity of immunotoxins is the dose-limiting factor in immunotoxin therapy. Which tissues are affected by nonspecific toxicity is dependent on the particular toxin used for immunotoxin preparation, and the ability of immunotoxins to penetrate into tissues and tumors is largely dependent on the size of the immunotoxins.
Large stable conjugated immunotoxins persist for long periods in blood vessels (T1/25-15 hour), thus endothelial cells are exposed to high toxin concentrations which may lead to endothelial cell damage. Smaller molecules, such as recombinant immunotoxins which rapidly leave the vascular system, would presumably have different toxicity. In humans, immunotoxins made with ricin and other ribotoxins, as well as with Pseudomonas exotoxin A (PE), Diphtheria toxin (DT) and their truncated derivatives have produced a variety of toxicities. These include vascular leak syndrome (mainly ricin immunotoxins) as well as liver toxicity (PE-derived immunotoxins). Vascular leak syndrome observed with ricin immunotoxins in animals and man may be explained by specific binding of ricin A-chain to endothelial cells and subsequent killing of the cells and damage to the vessels. The nonspecific liver-toxicity of PE immunotoxins is likely to be due to easy access and very rapid nonspecific uptake and internalization of proteins by hepatocytes. However, it is also possible that PE contains, in addition to the specific cell-binding site (Domain I) which is removed in most immunotoxins, an additional site which could be recognized with low affinity by hepatocytes, thus accounting for liver toxicity.
Another major impediment with immunotoxins in their clinical application is the human immune response against them, mainly toward the toxin moiety. Bacterial toxins like PE and DT are highly immunogenic and cannot be humanized with standard techniques. Usage of DT-derived immunotoxins is limited because most people in developed countries have been vaccinated against DT and many adults have neutralizing antibodies to DT. Immunogenicity is a problem to which so far no practical solution has been found. Reduced immunogenicity of these molecules would greatly improve the clinical application of immunotoxins.
An example of the successful use of an immunotoxin is the elimination of activated T cells which express high affinity IL2 receptors (IL2R), whereas normal resting T cells and their precursors do not. An immunotoxin made of IL2 could theoretically eliminate IL2R-expressing leukemia cells or IL2R-expressing immune cells involved in various disease states while not destroying IL2R negative normal cells, thereby preserving the full repertoire of antigen receptors required for T cell immune responses.
A chimeric protein, IL2-PE40, was produced and shown to eliminate activated T cells (Lorberboum-Galski et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:1922). IL2-PE40 was extremely cytotoxic to IL2R-expressing cell lines of human, ape and murine origin. It was also extremely cytotoxic to Con A-stimulated mouse and rat spleen cells, and had a suppressive effect against antigen-activated mouse cells and the generation of cytotoxic T cells in mixed lymphocyte cultures (Lorberboum-Galski et al., 1988, J. Biol. Chem. 263:18650-18656; Ogata et al., 1988, J. Immunol. 41:4224-4228; Lorberboum-Galski et al., 1990, J. Bio. Chem. 265:16311-16317).
A highly purified IL2-PE40 preparation (Bailon et al., 1988, Biotechnol. 6:1326-1329) was shown to (a) delay and mitigate adjuvant induced arthritis in rats (Case et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:287-291), (b) significantly prolong the survival of vascularized heart allograft in mice (Lorberboum-Galski et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:1008-1012) and corneal allografts in rats (Herbort et al., 1991, Transplant. 52:470-474), (c) reduce the incidence and severity of experimental autoimmune uveoretinitis in rats (Roberge et al., 1989, J. Immunol. 143:3498-3502), (d) suppress the growth of an IL2R bearing T cell lymphoma in mice (Kozak et al., 1990, J. Immunol. 145:2766-2771) and (e) prevent the development of experimental allergic encephalomyelitis, a T cell mediated disease of the central nervous system, in rats and mice (Beraud et al., 1991, Cell. Immunol. 133:379-389; Rose et al., 1991, J. Neuroimmunol. 32:209-217). However, such immunotoxin still suffers from the same deficiencies outlined above, particularly non-specific toxicity and immunogenicity in the human host.
2.2. APOPTOSIS-INDUCING PROTEINS
The development of multilineage organisms and the maintenance of homeostasis within tissues both require tightly regulated cell death. The ability of an individual cell to execute a suicidal response following a death stimulus varies markedly during its differentiation. Both positive and negative regulators of programmed cell death (apoptosis) have been identified.
A high percentage of follicular lymphomas have a characteristic chromosomal translocation, which places the proto-oncogene, Bcl-2 next to the immunoglobulin heavy chain locus, resulting in deregulation of Bcl-2 expression. Bcl-2 was found to function as a repressor of programmed cell death (Vaux et al., 1988, Nature 334:440-442). Recently, other Bcl-2 homologues were shown to inhibit apoptosis. However, one such homologue, Bax, mediates an opposite effect by accelerating apoptosis. An expanding family of Bcl-2 related proteins has recently been noted to share homology that is principally, but not exclusively, clustered within two conserved regions known as Bcl-2 homology domains 1 and 2 (BH1 and BH2) (Oltvai et al., 1993, Cell 74:609-619; Boise et al., 1993, Cell 74:597-608; Kozopas et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:3516-3520; Lin et al., 1993, J. Immunol. 151:1979-1988). Members of the Bcl family include Bax, Bcl-XL, Mcl-1, A1 and several open reading frames in DNA viruses. Another conserved domain in Bax, distinct from BH1 and BH2 was identified and termed BH3. This domain mediates cell death and protein binding functions (Chittenden et al., 1995, EMBO J. 14:5589-5596). Another member of the pro-apoptotic proteins contains only the BH3 domain, implying that this particular domain may be uniquely important in the promotion of apoptosis (Diaz et al., 1997, J. Biol. Chem. 272:11350-11355).
Bax homodimerizes and forms heterodimers with BCL-2 in vivo. Overexpressed Bax overcomes the death repressor activity of Bcl-2 (Oltvai et al., 1993, Cell 74:609-619). It was found that levels of Bax expression higher than Bcl-2 in bladder tumors was correlated with a better outcome for patients. Early relapses were much more frequently observed in patients whose tumors expressed more Bcl-2 than Bax mRNA (Gazzaniga et al., 1996, Int. J. Cancer 69:100-104).
Recently it was reported that Bax-alpha, a splice variant of Bax was expressed in high amount in normal breast epithelium, whereas only weak or no expression could be detected in 39 out of 40 cancer tissue samples examined (Bargou et al., 1996, J. Clin. Invest. 97:2651-2659). Of interest, downregulation of Bax-alpha was found in different histological subtypes. Furthermore, when Bax-alpha was transfected into breast cancer cell lines under the control of a tetracycline-dependent expression system, Bax restored sensitivity of the cancer cells toward both serum starvation and APO-I/Fas-triggered apoptosis, and significantly reduced tumor growth in SCID mice. Therefore, it was proposed that dysregulation of apoptosis might contribute to the pathogenesis of breast cancer at least in part due to an imbalance between members of the Bcl-2 gene family (Bargou et al., 1996, J. Clin. Invest. 97:2651-2659).
In another study, the expression of Bax was investigated in 52 cases of Hodgkin""s disease in parallel with Epstein-Barr virus, and was compared with the immunodetection of other apoptosis-regulating proteins, Mcl-1, Bcl-2 and Bcl-x. Bax expression was frequently detected in Hodgkin""s disease, providing an explanation for the good chemoresponses generally obtained for patients with this neoplastic disorder (Rigal-Haguet et al., 1996, Blood 87:2470-2475).
Additional members of this growing family of apoptosis inducing proteins have been cloned and identified. Bak is a new member of the Bcl-2 family which is expressed in a wide variety of cell types and binds to the Bcl-2 homologue Bcl-x2 in yeast (Farrow et al., 1995, Nature 374:731-733; Chittenden et al., 1995, Nature 374:733). A domain in Bak was identified as both necessary and sufficient for cytotoxicity activity and binding to Bcl-xl. Sequences similar to this domain that are distinct from BH1 and BH2 have been identified in Bax and Bip1. This domain was found to be of central importance in mediating the function of multiple cell death-regulatory proteins that interact with Bcl-2 family members (Chittenden et al., 1995, EMBO J. 14:5589-5596).
Overexpression of Bak in sympathetic neurons deprived of nerve growth factor accelerated apoptosis and blocked the protective effect of co-injected E1B 19K. The adenovirus E1B 19K protein is known to inhibit apoptosis induced by E1A, tumor-necrosis factor-alpha, FAS antigen and nerve growth factor deprivation (Farrow et al., 1995, Nature 374:731-733). Expression of Bak induced rapid and extensive apoptosis of serum-deprived fibroblasts, thus raising the possibility that Bak is directly involved in activating the cell death machinery (Chittenden et al., 1995, Nature 374:733-736). It was also reported that in the normal and neoplastic colon, mucosal expression of immunoreactive Bak co-localized with sites of epithelial cell apoptosis. Induction of apoptosis in the human colon cancer cell line HT29 and the rat normal small intestinal cell line 1EC 18 in culture was accompanied by increased Bak expression without consistent changes in expression of other Bcl-2 homologous proteins (Moss et al., 1996, Biochem. Biophys. Res. Commun. 223:199-203). Therefore, Bak was also suggested to be the endogenous Bcl-2 family member best correlated with intestinal cell apoptosis (Moss et al., 1996, Biochem. Biophys. Res. Commun. 223:119-203).
Unlike Bax, however, Bak can inhibit cell death in an Epstein-Barr-virus-transformed cell line. Tissues with unique distribution of Bak messenger RNA include those containing long-lived, terminally differentiated cell types (Krajewski et al., 1996, Cancer Res. 56:2849-2855), suggesting that cell-death-inducing activity is broadly distributed, and that tissue-specific modulation of apoptosis is controlled primarily by regulation of molecules that inhibit apoptosis (Kiefer et al., 1995, Nature 374:736-739).
Another member of the Bcl2 family is Bad that possesses the key amino acid motifs of BH1 and BH2 domains. Bad lacks the classical C-terminal signal-anchor sequence responsible for the integral membrane positions of other family members. Bad selectively dimerizes with Bcl-xL as well as Bcl-2, but not with Bax, Bcl-Xs-Mcl1, A1 or itself. Bad reverses the death repressor activity of Bcl-XL, but not that of Bcl-2 (Yang et al., 1995, Cell 80:285-291; Ottilie et al., 1997, J. Biol. Chem. 272:30866-30872; Zha et al., 1997, J. Biol. Chem. 272:24101-24104).
Another member is Bik which interacts with the cellular survival-promoting proteins, Bcl-2 and Bcl-XL as well as the viral survival-promoting proteins, Epstein Barr virus-BHRF1 and adenovirus E1B-19 kDa. In transient transfection assays, Bik promotes cell death in a manner similar to other death-promoting members of the Bcl-2 family, Bax and Bak. This death-promoting activity of Bik can be suppressed by coexpression of Bcl-2, Bcl-XL, EBV-BHRF1 and E1B-19 kDa proteins suggesting that Bik may be a common target for both cellular and viral anti-apoptotic proteins. While Bik does not contain overt homology to the BH1 and BH2 conserved domains characteristic of the Bcl-2 family, it shares a 9 amino acid domain (BH3) with Bax and Bak which may be a critical determinant for the death-promoting activity of these proteins (Boyd et al., 1995, Oncogene 11:1921-1928; Han et al., 1996, Mol. Cell. Biol. 16:5857-5864).
The Bcl-2 family is composed of various pairs of antagonist and agonist proteins that regulate apoptosis. Whether their function is interdependent is uncertain. Using a genetic approach to address this question, Knudson et al. (1997, Nature Genetics 16:358-363), recently utilized gainxe2x80x94and loss ofxe2x80x94function models of Bcl-2 and Bax, and found that apoptosis and thymic hypoplasia, characteristic of Bcl-2-deficient mice, are largely absent in mice also deficient in Bax. A single copy of Bax promoted apoptosis in the absence of Bcl-2. In contrast, overexpression Bcl-2 still repressed apoptosis in the absence of Bax. While an in vivo competition exists between Bax and Bcl-2, each is able to regulate apoptosis independently. Bax has been shown to form channels in lipid membranes and trigger the release of liposome-encapsulated carboxyluorescein at both neutral and acidic pH. At physiological pH, release could be blocked by Bcl-2. In planer lipid bilayers, Bax formed pH- and voltage-dependent ion-conduction channels. Thus, the pro-apoptotic effects of Bax may be elicited through an intrinsic pore-forming activity that can be antagonized by Bcl-2 (Antonsson et al., 1997, Science 277:370-372). Two other members of this family, Bcl-2 and Bcl-1, were also shown to form pores in lipid membranes (Schendel et al., 1997, Proc. Natl. Acad. Sci. U.S.A. 94:5113-5118).
Prior to the present invention, a fusion protein containing a Bcl-2 pro-apoptotic member was not reported, nor was it predictable if such a molecule could retain biological activites when added to a cell exogenously to induce apoptosis.
The present invention relates to chimeric proteins with cell-targeting specificity and apoptosis-inducing activities. The chimeric proteins of the invention are composed of a cell-specific targeting moiety and an apoptosis-inducing moiety. The cell-specific targeting moiety provides cell-specific binding properties to the chimeric protein, while the apoptosis-inducing moiety induces programmed cell death upon entry into a target cell. It is preferred that the chimeric proteins of the invention be produced by recombinant expression of a fusion polynucleotide between a coding sequence of a cell-targeting moiety and a coding sequence of an apoptosis-inducing protein. Such chimeric proteins are likely to be superior to the immunotoxins currently used in the art because they are of human origin and thus are expected to have reduced immunogenicity in a human recipient. In addition, chimeric proteins kill target cells by inducing apoptosis which does not cause a release of cellular organelles into the extracellular environment to result in an inflammatory response. When cells die by the apoptotic pathway, they shrink and condense, but the organelles and plasma membranes retain their integrity, and the dead cells are rapidly phagocytosed by neighboring cells or macrophages before there is leakage of the cells"" contents, thereby eliciting minimal tissue or systemic response.
The invention also relates to pharmaceutical compositions of the chimeric proteins, methods of producing such proteins, and methods of using the same in vitro and in vivo, especially for eliminating specific undesirable target cells, and for the treatment of a variety of disease conditions as well as the use of the proteins for disease diagnosis.
The invention is based, in part, on the Applicants"" discovery that a partially purified recombinant chimeric protein, IL2-Bax, specifically targets IL2R+ cells, which include but are not limited to, T. cells, B cells, monocytes and natural killer cells. The protein kills target cells by inducing apoptosis of these cells. A wide variety of uses are encompassed by the present invention, including but not limited to, the treatment of autoimmunity, transplantation rejection, graft-versus-host disease, cancer, hypersensitivity, and infectious diseases.